Research ArticleFatty Acid Profiles of Stipe and Blade from the NorwegianBrown Macroalgae Laminaria hyperborea with SpecialReference to Acyl Glycerides, Polar Lipids, and Free Fatty Acids
Lena Foseid, Hanne Devle, Yngve Stenstrøm,Carl Fredrik Naess-Andresen, and Dag Ekeberg
Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, 1432 As, Norway
Correspondence should be addressed to Hanne Devle; [email protected]
Received 31 December 2016; Revised 7 May 2017; Accepted 24 May 2017; Published 20 June 2017
A thorough analysis of the fatty acid profiles of stipe and blade from the kelp species Laminaria hyperborea is presented. Lipidextracts were fractionated into neutral lipids, free fatty acids, and polar lipids, prior to derivatization and GC-MS analysis. A totalof 42 fatty acids were identified and quantified, including the n-3 fatty acids 𝛼-linolenic acid, stearidonic acid, and eicosapentaenoicacid. The fatty acid amounts are higher in blade than in stipe (7.42mg/g dry weight and 2.57mg/g dry weight, resp.). The highestamounts of n-3 fatty acids are found within the neutral lipid fractions with 590.6 ug/g dry weight and 100.9 ug/g dry weight forblade and stipe, respectively. The amounts of polyunsaturated fatty acids are 3.4 times higher in blade than stipe. The blade had thehighest PUFA/SFA ratio compared to stipe (1.02 versus 0.76) and the lowest n-6/n-3 ratio (0.8 versus 3.5). This study highlights thecompositional differences between the lipid fractions of stipe and blade from L. hyperborea. The amount of polyunsaturated fattyacids compared to saturated- and monounsaturated fatty acids is known to influence human health. In the pharmaceutical, food,and feed industries, this can be of importance for production of different health products.
1. Introduction
The increase in world population and lack of sufficient foodbeg for new sources of food and feed. As much as 60% of theworld food energy intake is provided by the cereals wheat,rice, and corn [1]. These cereals, while high in metabolizableenergy and carbohydrates, have small amounts of importantnutrients such as proteins, minerals, vitamins (especially Aand C), and fatty acids, especially long chained polyunsatu-rated n-3 fatty acids [2–4]. A promising supplement for foodand feed is a better utilization ofmarine resources.World pro-duction and harvesting of micro- and macroalgae have dou-bled from 2004 to 2014 [5]. Still, 97% of the production andharvesting is found in Asia [5]; thus, there is a large potentialfor expansion in other parts of the world. Macroalgae area diverse group of marine plants, informally divided intothree groups: Rhodophyta (red algae), Chlorophyta (greenalgae), and Phaeophyta (brown algae). Use of seaweed as feed,food, and fertilizers in times of food shortage was common
in northern Europe from around the 10th century and upuntil about middle of the 18th century [6]. At present timein Scandinavia and other Western countries, the utilizationof seaweeds is limited to industrial products, such as alginate,agar, carrageenan, and thickeners, and only scarcely used infood and feed industries. Biomarine processing industrieshave great potential in coastal regions. Norway is particularlyprivileged due to the long coastline combined with thepresence of nutritious ocean currents (North Atlantic Driftand Norwegian Coastal Current), which ensure a goodclimate for growth of marine flora and fauna.
Seaweed has for many years been thought to havepositive effect on human health, and consumption of thesemarine plants has been linked to a lower incidence ofcancer, hyperlipidemia, and coronary heart disease [7]. Theyare also reported to possess antimicrobial, antiviral, anti-inflammatory, and immunotropic properties [8]. Many of thereportedmedicinal effects ofmarine algae have not been con-firmed, but Brown et al. and Stein and Borden have published
HindawiJournal of LipidsVolume 2017, Article ID 1029702, 9 pageshttps://doi.org/10.1155/2017/1029702
comprehensive reviews on this topic [7, 9]. Lipid profiles andcompositions can be of importance for human health and forcommercial application [10–12]. Eukaryotic algae contain adiverse composition of acyl lipids and their fatty acids, albeitthe number of algae which have been comprehensively stud-ied is relatively few [11]. Lipids in macroalgae can be dividedinto neutral lipids that include monoglycerides, diglycerides,triglycerides, sterols, and polar lipids that include glycolipids,phospholipids, and betaine lipids [10, 11, 13]. An importantnutritional benefit of marine macroalgae is attributed to thehigh level of polyunsaturated fatty acids (PUFAs), especiallyn-3 and n-6 fatty acids [14]. A diet with a low n-6/n-3ratio is reported to have suppressive effects on cardiovasculardiseases, cancer, and inflammatory and autoimmune diseases[15]. With a global decline in fish stocks, seaweed may bea good option for an alternative and sustainable source ofn-3 PUFAs [7]. Previous studies regarding fatty acids inbrown macroalgae have had a nutritional or pharmaceuticalfocus. Fatty acid content has only been determined as oneamong several parameters, resulting in limited fatty acidprofiles [16–20]. More extensive FA profiling has been doneon certain species of brown macroalgae [8, 21–23]. Little ishowever known about fatty acid compositions of individuallipid classes of marinemacrophytes in general [8].The brownmacroalgae Laminaria hyperborea, a common species of kelpfound in the northern Atlantic, has only previously beencharacterized with regard to fatty acids in three studies [6,12, 24], all presenting limited profiles identifying no morethan nine fatty acids. Within-plant fatty acid distribution forthis species has previously only been reported by Schmidand Stengel [24] with Laminaria hyperborea harvested onthe west coast of Ireland. Variations in distribution of fattyacids between different parts of the seaweed depend on themorphology of the species as well as its physiological andecological circumstances [25].
Before considering the marine algae as a food source, it isimportant to assess its nutritional value. In this context, theaim of this study has been to provide a thorough analysis ofthe fatty acid profiles in stipe and blade from the macroalgaeL. hyperborea. In this study, the lipids were fractionated intofree fatty acids (FFAs), acyl glycerides (neutral lipids, NLs),and polar lipids (PLs) by using solid phase extraction. Thefatty acids from each class were identified and quantified byGC-MS.
2. Material and Methods
Chloroform and hexane were of Chromasolv� quality, hep-tane, diethyl ether, methanol, and NaCl puriss pa. quality, allfrom Sigma-Aldrich (St. Louis, MO, USA). The acetic acidwas from Honeywell Riedel-de Haen (Seelze, Germany).
2.1. Standards. A fatty acid methyl ester (FAME) mix with 37components (Food Industry FAME MIX, Restek, Bellefonte,PA, USA) was used for identification of the FAMEs. A21-component FAME mix (Qualmix PUFA Fish M, MethylEsters (Menhaden Oil), Larodan AB, Solna, Sweden) wasused for identification of all-cis-6,9,12,15-octadecatetraenoicacid methyl ester, all-cis-8,11,14,17-eicosatetraenoic acid
methyl ester, and all-cis-7,10,13,16,19-docosapentaenoic acidmethyl ester. In addition, the following individual FAMEstandards were used: nonanoic acid methyl ester (Sigma-Aldrich, St. Louis, MO, USA), 13-methyltetradecanoicacid methyl ester, trans-9-tetradecenoic acid methyl ester,cis-9-heptadecenoic acid methyl ester, cis-13-octadecenoicacid methyl ester, cis-9-eicosenoic acid methyl ester, andhexacosanoic acid methyl ester (all from Larodan AB, Solna,Sweden). Three internal standards were used (10mg/mL,dissolved in CHCl
3), one for each lipid fraction; 1,2-
dinonadecanoyl-sn-glycero-3-phosphatidylcholine for thePL fractions, nonadecanoic acid for the FFA fractions, andtrinonadecanoin for the NL fractions (all from Larodan AB,Solna, Sweden). Nonadecanoic acid methyl ester (LarodanAB, Solna, Sweden) was added to the 37 components FAMEmix for retention time identification, since C19:0 was used asinternal standard in the samples.
2.2. Pretreatment of L. hyperborea. L. hyperborea was pro-vided and identified by FMC BioPolymer AS. It was har-vested off the west coast of Norway, outside Sør-TrøndelagCounty Municipality in October 2015. On board the trawlerL. hyperborea was rinsed, crude-cut, and preserved withformalin manually. The holdfast was discarded. Once offthe trawler stipe and blade were vacuum packed separately.When received at the university, stipe and blade were rinsedwith water to eliminate contaminants, frozen with liquid N
2
(99.999%, AGA, the Linde Group, Munich, Germany), andfreeze-dried (Alpha 2–4 LDplus,Martin Christ Gefriertrock-nungsanlagen GmbH, Osterode am Harz, Germany). Thefreeze-dried material was crushed in a QMM Micromixerand pulverized in a Laboratory Mixer 3100 (Danfoss) by G.A. Lund at Pharmatech AS, Fredrikstad, Norway. The watercontent of fresh seaweed was measured according to ISO11465:1993/Cor1:1994.
2.3. Lipid Extraction. Four sample replicates of both stipe andblade were used and they were all treated separately duringthe sample preparation stages. The lipids were extracted witha modified Folch’s method [26]. In brief, 5–10 g alga powderwas extracted in a separatory funnel with 10 times its volumeCHCl
3:MeOH (2/1), and 50𝜇L of each internal standard was
added with a Hamilton� syringe. To induce phase separation,0.9% NaCl was added after mixing (0.2 times the volumeof CHCl
3:MeOH). After approximately 20min the organic
phase was transferred to a test tube (Duran� 20 × 150mm,Mainz, Germany). The polar phase was reextracted withCHCl
3, 30–60mL depending on amount of alga powder.The
organic phases of each samplewere combined and evaporatedwith a vacuum evaporator (Q-101, Buchi Labortechnik AG,Flawil, Switzerland) at 40∘C, redissolved in 1.00mL chloro-form, and transferred to vials for SPE.
A liquid-handling robot (Gilson, GX-271, ASPEC, Mid-dleton, WI, USA) was used to carry out the SPE procedure.The method used was based on previous work by Pinkart etal. 1998 and Ruiz et al. 2004 [27, 28]. Aminopropyl-modifiedsilica phase SPE columns, 500mg, 3mL (Chromabond,Macherey-Nagel, Duren, Germany) were conditioned with7.5mL hexane before 500 𝜇L of sample was applied. The
Journal of Lipids 3
NLs (mono-, di-, and triglycerides) were eluted with 5mLchloroform, then the FFAs with 5mL diethyl ether:aceticacid (98 : 2 v/v), and lastly the PLs with 5mL methanol. Thepossibility of cross contaminations between any of the threeclasses was checked by performing tests with standards foreach lipid class. The recovery was 90% or higher. The eluateswere transferred to culture tubes (Duran 12× 100mm,Mainz,Germany) and evaporated under N
2(g) at 40∘C.
2.4. Formation of FAMEs. For formation of FAMEs the NLand PL fractions were redissolved in 2mL of heptane, beforeaddition of 1.5mL of 3.3mg/mL sodium methoxide. Thesodium methoxide solution was made by dissolving metallicsodium (Purum, Merck, Darmstadt, Germany) in methanolto a concentration of 3.3mg/mL. The culture tubes werethen shaken horizontally for 30min at 350 rpm (Biosan Ltd.,PSU-10i, Riga, Latvia) and left to settle vertically for 10minbefore the heptane phases were transferred to vials for storageat −20∘C prior to GC-MS analysis. The FFA fractions wereredissolved in 1mL BF
3-MeOH (14%, Sigma-Aldrich, St.
Louis, MO, USA).The samples were heated for 5min at 70∘Cin awater bath. After heating, 1mLheptanewas added to eachsample tube before mixing on a vortex mixer. The heptanephases were transferred to vials and stored at −20∘C prior toanalysis by GC-MS.
2.5. Analysis of FAMEs byGC-MS. Theanalysis of the FAMEswas based on a previously published method [29]. Shortly,the analyses were carried out on an Agilent 6890 Seriesgas chromatograph (GC; Agilent Technology, Wilmington,DE, USA) in combination with an Autospec Ultima massspectrometer (MS; Micromass Ltd., Manchester, England)using an EI ion source.TheGCwas equippedwith aCTCPALAutosampler (CTC Analytics, AG, Zwingen, Switzerland).Separation was carried out on a 60m Restek column (Rtx�-2330) with 0.25mm ID and a 0.2𝜇m film thickness of fusedsilica 90% biscyanopropyl/10%phenylcyanopropyl polysilox-ane stationary phase (Restek Corporation, Bellefonte, PA,USA). For carrier gas, helium (99.99990%, fromYara, Rjukan,Norway) was used at 1mL/min constant flow. The EI ionsource was used in positive mode, producing 70 eV electronsat 250∘C.TheMS was scanned in the range 40–600m/z with0.3 s scan time, 0.2 s interscan delay, and 0.5 s cycle time. Thetransfer line temperature was set at 270∘C.The resolution was1000.
A split ratio of 1/10 was used with injections of 1.0 𝜇Lsample. Two injections parallels were used for each samplereplicate. Identification of fatty acids was performed bycomparing retention times with standards as well as MSlibrary searches. MassLynx version 4.0 (Waters, Milford,MA,USA) and NIST 2014 Mass Spectral Library (Gaithersburg,MD, USA) were used. Relative response factors previouslydetermined by Devle et al. [29] were employed for quan-titative determination. The resulting amounts are given in𝜇g/g dry weight (DW). The GC oven had a start temperatureof 65∘C, held for 3min, the temperature was then raised to150∘C (40∘C/min), held for 13min, before being increasedto 151∘C (2∘C/min), held for 20min, with a slow increase to230∘C (2∘C/min), and held for 10min, before a final increase
to 240∘C (50∘C/min), and the end temperature was held for3.7min.
3. Results and Discussion
We have identified and quantified 42 different fatty acids inL. hyperborea, as shown in Table 1. This is a significantlyhigher number than previously reported by others for thisspecies [6, 12, 24]. Seaweeds usually contain a lipid level of<1–5% [19, 21, 30].The portions of the total lipids that containmolecules with fatty acids depend significantly on species,geographical location, and seasonal changes [12, 18, 24, 31, 32].In our study, the total FA (TFA) content relative to dry weightin blade and stipe was 0.74% and 0.26%, respectively.This 3 : 1ratio between blade and stipe is consistent with Schmid andStengels [24] findings of within-plant variations for the samespecies, even though they had twice the TFA content (0.5%and 1.5% in stipe and blade, resp.). The water content wasfound to be 83.3% ± 0.5 and 85.6% ± 0.8 in blade and stipe,respectively.The fatty acid profile was determined for the NL,FFA, and PL fractions in stipe and blade separately. For theindividual lipid fractions, the %TFA was the highest in NLswith 42.9% and 54.5% in stipe and blade, respectively. ThePL fraction for the stipe consisted of 48.5% TFA versus 31.5%TFA in blades. The %TFA for the FFAs ranged from 8.6% instipe to 13.9% in blade. While up to 41 different fatty acidswere detected within a lipid fraction, the same 10 fatty acidspredominated in all fractions in both stipe and blade. In thisgroup of 10, three FAs were saturated (SFA, C14:0, C16:0, andC18:0), one was monounsaturated (MUFA, C18:1 cis-9) andthe remaining five were polyunsaturated (PUFA, C18:2 cis-9,12, C18:3 cis-9,12,15, C18:4 cis-6,9,12,15, C20:4 cis-5,8,11,14,and C20:5 cis-5,8,11,14,17).
The predominating fatty acids constitute more than 90%of the total fatty acid content in all the fractions, as shownin Figure 1. They are found in amounts varying from 0.65to 1200𝜇g/g DW (Table 1). A fatty acid was classified aspredominating if it was above 2%of the total fatty acid contentin at least one of the lipid fractions in either stipe or blade.All these ten fatty acids corresponded to those identified byothers [6, 12, 24]. Schmid and Stengel [24] identified C18:3n-6, at 1.2% in stipe and 5.5% in blade, which differs fromour results, where C18:3n-6 is not above 2% in any of theblade lipid fractions.This could be due to geographical and/orseasonal variations. The same FA was not detected at all byVan Ginneken et al. [12] and also not reported by Mæhreet al. [6], who both studied L. hyperborea as one of severalmacroalgal species. Only a maximum of two trans fatty acids,C14:1 trans-9 and C16:2 cis or trans-7,10, were identified inthe samples, both in relatively low amounts (≤2.53 𝜇g/g DW).Among the predominating fatty acids are important dietaryn-3 fatty acids such as 𝛼-linolenic acid (ALA, C18:3n-3),stearidonic acid (SDA, C18:4n-3), and eicosapentaenoic acid(EPA, C20:5n-3) as well as two n-6 fatty acids, linoleic acid(LA, C18:2n-6),and arachidonic acid (AA, C20:4n-6). Howfavorable L. hyperborea is for the human diet (and thus also inanimal feed) depends on several factors, for example, contentof essential FAs (LA and ALA), other important nutritional
4 Journal of Lipids
Table1:Fatty
acid
contentinthelipid
fractio
ns(m
ean±SD
,𝜇g/gDW)o
fstip
eand
bladefrom
L.hyperborea
(𝑛=4,twoinjectionparallelsfore
achof
thesefou
rsam
plingevents)
.
Fatty
acid
Stipe
Blade
NL
FFA
PLNL
FFA
PLC7
:05methyla
1.32±0.09
n.d.
n.d.
0.49±0.03
n.d.
n.d.
C8:0
1.47±0.11
0.70±0.04
n.d.
5.36±0.63
0.65±0.03
n.d.
C9:0
n.d.
0.41±0.06
n.d.
n.d.
0.64±0.03
n.d.
C10:0
0.76±0.05
n.d.
n.d.
4.98±0.63
0.39±0.06
n.d.
C12:0
0.53±0.09
1.66±0.22
0.13±0.01
0.73±0.03
2.87±0.12
0.09±0.01
C13:0
n.d.
0.19±0.01
n.d.
0.17±0.01
0.96±0.03
0.07±0.01
C14:0
101.2
8±1.8
021.94±0.78
272.39±9.9
2223.80±4.65
94.20±4.02
312.39±5.60
C14:013
methyl
n.d.
0.33±0.05
n.d.
n.d.
0.39±0.02
n.d.
C14:1trans-9
n.d.
0.20±0.02
n.d.
n.d.
1.86±0.12
n.d.
C14:1cis-9
0.89±0.04
0.13±0.02
0.27±0.03
2.45±0.03
0.46±0.02
0.38±0.01
C15:0
0.95±0.05
1.00±0.05
2.52±0.03
8.73±0.18
6.40±0.18
10.43±0.14
C16:0
163.36±5.08
82.19±3.08
276.64±6.91
900.21±17.85
298.88±12.12
417.2
9±5.39
C16:1b
n.d.
n.d.
n.d.
0.77±0.10
1.31±
0.03
0.65±0.02
C16:1b
n.d.
0.53±0.04
0.37±0.05
2.24±0.09
2.75±0.09
1.05±0.02
C16:1cis-9
74.99±2.56
14.38±0.21
54.60±1.0
2179.6
5±1.9
474.12±2.11
104.78±1.14
C17:0
0.32±0.02
0.52±0.06
0.57±0.04
6.62±0.24
1.73±0.07
1.83±0.05
C16:2cis
ortra
ns-7,10
(n-6)
0.62±0.04
0.13±0.02
0.49±0.07
2.00±0.07
1.72±0.05
2.53±0.05
C17:1cis-9
0.33±0.03
0.10±0.04
0.35±0.3
4.14±0.24
2.02±0.05
1.96±0.06
C18:0
5.95±0.28
17.35±0.71
2.54±0.09
84.00±2.01
21.04±0.70
4.42±0.06
C18:1cis-9
234.12±5.87
47.46±0.43
418.07±10.33
1200.51±
29.95
219.9
0±5.17
546.84±11.59
C18:1cis-11
3.3 7±0.31
2.27±0.04
4.85±0.22
11.39±0.21
6.64±0.14
7.68±0.14
C18:2all-cis-9,1
2(LA)c
(n-6)
44.67±1.7
62.70±0.09
48.39±101
183.66±4.19
27.79±0.67
88.57±1.19
C18:3all-cis-6,9,1
2(n-6)
3.23±0.22
n.d.
3.59±0.13
14.02±0.18
3.02±0.06
12.29±0.19
C20:0
6.67±0.54
1.35±0.07
1.52±0.10
49.59±1.19
9.80±0.25
4.79±0.09
C18:3all-cis-9,1
2,15(A
LA)d
(n-3)
7.31±
0.31
0.65±0.03
2.37±0.15
83.56±1.3
821.99±0.53
25.22±0.68
C20:1cis-9
n.d.
0.39±0.08
3.34±0.18
7.87±0.40
4.17±0.05
9.75±0.19
C20:1cis-11
n.d.
0.16±0.04
0.39±0.05
0.89±0.05
0.39±0.02
0.95±0.03
C18:4all-cis-6,9,1
2,15(SDA)e
(n-3)
5.6±0.3
0.93±0.03
10.74±0.24
68.90±1.2
964
.62±1.3
3259.5
8±2.77
C20:2all-cis-11,14
(n-6)
3.73±0.35
0.27±0.05
4.88±0.18
10.35±0.33
2.87±0.12
8.57±0.17
C20:3b
1.93±0.16
0.12±0.02
0.90±0.07
1.91±
0.05
0.50±0.03
1.08±0.03
C20:3b
1.49±0.14
0.07±0.01
1.04±0.06
4.50±0.59
0.72±0.04
2.71±0.05
C20:3b
n.d.
0.04±0.01
0.43±0.03
1.20±0.63
0.28±0.02
0.79±0.03
C20:3all-cis-8,11,14
(n-6)
3.63±0.35
0.16±0.02
5.19±0.20
18.23±1.2
90.80±0.02
4.46±0.08
C22:0
n.d.
0.06±0.01
n.d.
0.93±0.13
0.41±0.02
n.d.
C20:4all-cis-5,8,11,14(A
A)f
(n-6)
344.56±11.46
17.39±0.39
82.89±1.4
9516.97±11.52
66.32±2.11
139.9
0±1.2
5C2
0:4b
n.d.
n.d.
0.44±0.04
2.80±0.14
0.92±0.04
3.21±0.07
C20:4all-cis-8,11,14
,17(n-3)
3.29±0.26
n.d.
2.39±0.13
13.52±0.33
3.76±0.17
10.12±0.17
C20:5all-cis-5,8,11,14
,17(EPA)g
(n-3)
81.72±3.28
2.91±0.07
40.62±0.87
409.2
7±9.5
181.58±1.9
0339.0
9±3.60
Journal of Lipids 5
Table1:Con
tinued.
Fatty
acid
Stipe
Blade
NL
FFA
PLNL
FFA
PLC2
4:0
n.d.
0.29±0.02
n.d.
0.64±0.08
1.45±0.06
n.d.
C22:5all-cis-7,10,13,16
,19(n-3)
3.01±0.28
0.18±0.01
1.56±0.09
15.37±0.50
4.97±0.12
11.29±0.26
C22:6all-cis-4,7,10,13,16
,19(D
HA)h
(n-3)
n.d.
0.08±0.01
0.33±0.06
n.d.
1.67±0.06
0.22±0.02
C26:0
n.d.
0.27±0.02
n.d.
n.d.
1.08±0.07
n.d.
Total
1101.14
219.5
11244
.77
4042.45
1033.02
2334.99
a Fattyacidsareidentifi
edby
NISTlib
rary
search
only;bun
know
niso
meroffatty
acid,identified
byNISTlib
rary
search
only;cLA
:linoleica
cid;
d ALA
:alpha
linolenicacid;eSD
A:stearidon
icacid;fAA:arachidon
icacid;gEP
A:eicosapentaenoica
cid;
h DHA:docosahexaeno
icacid;n
.d.=
notd
etected.NL,neutrallipid;FFA
,freefattyacid;P
L,po
larlipid.
6 Journal of Lipids
Table 2: Sum of SFAs, MUFAs, and PUFAs, as well as n-6 and n-3 in L. hyperborea given in 𝜇g/g DW, (𝑛 = 4, two injection parallels for eachsample replicate).
Figure 1: Fatty acid profile for fatty acids contributingmore than 2%of total fatty acid content, in at least one lipid fraction. SUM < 2%is the summarized contribution of the remaining fatty acids (𝑛 = 4,two injection parallels for each of these four sample replicates, errorbars = ±SD). NL, neutral lipid; FFA, free fatty acid; PL, polar lipid.
FAs (SDA, AA, and EPA), and the ratios between PUFA/SFAand n-6/n-3 fatty acids.
It is known that brown seaweeds grown in temperate orsubarctic areas can accumulate n-3 and n-6 PUFAs [33]. Inboth stipe and blade the lowest amounts of n-3 and n-6 fattyacids are found in the FFA fraction. The highest amounts arefound in the NL fraction, with the exception of n-3 in bladewhere the amount in the PL fraction is higher than in the NLfraction (646 and 591𝜇g/gDW, resp.).Thehighest amounts ofthe essential FAs LA and ALA were found in the NLs for theblade (183.7 𝜇g/g DW and 83.6 𝜇g/g DW, resp.). However, forstipe, the highest amount of LA was found in the PL fraction
(48.4 𝜇g/mL) and highest amount of ALA was found in theNL fraction (7.3 𝜇g/g DW). There was a lower proportionof the n-6 FA arachidonic acid in blade versus stipe (9.8%and 17.3%, resp.). This corresponds to what was reported bySchmid and Stengel [24].
The World Health Organization (WHO) recommends adaily intake of 0.25 g EPA+ C22:6 n-3 (DHA) as part of ahealthy diet [34]. Even though seaweed can have high levelsof EPA, the fatty acid DHA is generally absent or only foundin small amounts in different phaeophytes [18]. The highestamount of DHA in our study was found in the blade FFAfraction (1.7 𝜇g/g DW). With a total content of EPA+DHA inblade at 832 𝜇g/g DW, achieving the recommended amountby consumption of seaweed alone is highly unlikely, as thedaily intake would have to be approximately 300 g driedseaweed (or 1500 g fresh seaweed). Although n-3 and n-6 PUFAs are usually easily oxidized, studies have shownthat these PUFAs have exhibited high oxidative stability inseaweed lipids in dried seaweed products [35].The reason forthismight be due to a protective effect of galactosyl and sulfo-quinovosyl moieties on PUFAs bonded to glycoglycerolipids(the main membrane lipids) [33].
Stipe and blade differ not only in content of the individualFAs but also in the amounts of SFAs, MUFAs, and PUFAs(Table 2). The stipe had the highest distribution of SFAwith 37.7% of TFA, whereas MUFA and PUFA for the stipewere at 33.6% and 28.7%, respectively. The blades, however,had a higher distribution of PUFAs with 34.2% of TFAand a lower distribution of SFA and MUFA with 33.4%and 32.4%, respectively. Stengel 2015 also reported a higherdistribution of PUFAs in blade versus stipe with 52.0% and32.2%, respectively. The FA distribution differs significantlywith geographical and seasonal variations, most likely due tonutrition, light conditions, and other biological factors. VanGinneken et al. [12] found a PUFA distribution of 53% of TFAin L. hyperborea (fronds) harvested in France with 25% SFAand 22%MUFA. Mæhre et al. [6] harvested the same species(whole plant) from the Norwegian coast and reported 34.2%PUFA, 33.7% SFA, and 26.5% MUFA. These results are verysimilar to our findings, even though there was a significantdistance in time of harvest (May/June 2010 versus October
Journal of Lipids 7
C14:
0
C16:
0
C16:
1 ci
s 9
C18:
0
C18:
1 ci
s 9
C18:
2 ci
s 9,1
2 (L
A)
C18:
3 ci
s 9,1
2,15
(ALA
)
C18:
4 ci
s 6,9
,12,
15 (S
DA
)
C20:
4 ci
s 5,8
,11,
14 (A
A)
C20:
5 ci
s 5,8
,11,
14,1
7 (E
PA)
2.2 5.
5
2.4
14.1
5.1
4.1 11
.4
12.3
1.5 5.
0
4.3
3.6 5.2
1.2 4.
6 8.4
33.9
69.3
3.8
28.0
1.1 1.5 1.9
1.7
1.3 1.8
10.7
24.2
1.7 8.
3
0
10
20
30
40
50
60
70
Am
ount
of b
lade
/am
ount
of s
tipe
NLFFAPL
Figure 2: Ratio between average blade and stipe amounts in the predominating fatty acids. NL, neutral lipid; FFA, free fatty acid; PL, polarlipid.
2015).The total amount of PUFAs in blade is 3.4 times higherthan in stipe, and the total amounts of SFAs and MUFAs are2.6 and 2.8 times higher, respectively. PUFAs are preferredover SFAs from a dietary perspective, and replacing SFAswith PUFAs in the diet decreases the risk of coronary heartdisease [34]. The NL fraction in stipe and the PL fractionin blade have the highest PUFA/SFA ratio of 1.79 and 1.21,respectively. When combining the lipid fractions the bladeFAs had a higher PUFA/SFA ratio compared to the stipe FAs(1.02 versus 0.76).
L. hyperborea has an n-6/n-3 ratio of 0.8/1 in blade and3.5/1 in stipe. The ratios vary between the lipid fractions, asseen in Table 2, but are higher in stipe than in blade. InWest-ern diet the n-6/n-3 ratio is 15–20/1 and this is significantlyhigher than ∼1, which was normal during human evolution[15, 36]. However, for health benefits lowering this ratio isconsidered to be beneficial and associated with preventionof inflammatory, cardiovascular, and neural disorders [12]. n-6/n-3 ratios of 2–5/1 are reported to have suppressive effectson cardiovascular, inflammatory, and autoimmune diseases[15, 37, 38]. Since there is a significant difference between then-6/n-3 ratios in stipe and blade, using only blade could beconsidered if a very low n-6/n-3 ratio is desired. Though, inthis context it should also be mentioned that FAO in their2010 report [34] do not consider this ratio to be importantand give no specific recommendations of such.
While the same fatty acids predominate, the amounts inblade are consistently higher than those in stipe, as seen inFigure 2. This is consistent with results found by Schmidand Stengel [24]. At minimum, the amounts in blade are 1.1times higher than in stipe for myristic acid (C14:0) in the PL
fraction, and at maximum, 69.3 times higher than in stipefor SDA in the FFA fraction. For the FFA and PL fractions,the largest differences are found in the fatty acids ALA, SDA,and EPA. In the NL fraction, the largest difference betweenstipe and blade amounts is found in stearic acid (C18:0), whileblade and stipe amounts are almost equal in the FFA and PLfractions for the same fatty acid.
4. Conclusions
A total of 42 different fatty acids are identified and quantifiedin the stipe and blade of L. hyperborea, with maximum twofatty acids having trans configuration. Some fatty acids arefound in either stipe or blade, while others are only presentin certain lipid fractions (NL, FFA, and PL) within stipe andblade. Among the predominating fatty acids are the n-3 fattyacids ALA (10.4 and 131 𝜇g/g DW), SDA (17.2 and 394 𝜇g/gDW), and EPA (126 and 830 𝜇g/g DW), as well as two n-6 fatty acids: LA (96 and 296𝜇g/g DW) and AA (444 and723 𝜇g/g DW); the values in parentheses are for stipe andblade, respectively.The ratios between n-6 and n-3 fatty acidsare ≤4.4/1 in all lipid fractions but especially low (≤1.3/1) inblade.The blades also presented the highest PUFA/SFA ratio.Regarding the potential of commercialization in respect ofnutritional applications of L. hyperborea, blade is found torepresent the most suitable material, due to higher levels ofPUFAs and a low n-6/n-3 ratio.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
8 Journal of Lipids
Acknowledgments
The authors would like to thank FMC Biopolymer forsampling the L. hyperborea and Pharmatech AS for millingall samples.The authors would also like to thank the InstituteofMarine Research for allowing them to use their picture of akelp forest in the graphical abstract.This work was supportedby the Norwegian University of Life Sciences.
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